Cancer is a leading cause of death in the United States with over 1,000,000 new cases being diagnosed every year. As a result of the high sensitivities and specificities that are required to justify large-scale population screening, only very few single protein biomarkers are routinely used today in the clinical setting. It is of critical priority to develop novel technologies that will enable the rapid detection of a plethora of biomarkers relevant to early diagnosis, prognosis, staging and treatment response. The long-term objective of this research is to combine the emerging technology of microfluidics with state-of-the-art mass spectrometry (MS) detection to enhance our capacity for analyzing molecular structure and function in biological systems. This application capitalizes on the distinguishing capabilities of microfluidic architectures that enable process integration, multiplexing, fast and high-throughput processing of minute amounts of sample, and the power of MS detection that provides the sensitivity, specificity and resolving power necessary for unambiguous detection of trace level components. Specific Aim 1: Develop a compact, low-cost and disposable microfluidic analysis platform with matrix assisted laser desorption ionization (MALDI)-MS/MS detection for high-throughput proteomics that will enable the study of protein co-expression patterns and biomarker discovery. The microfluidic device will carry out parallel liquid chromatography (LC) separations and will integrate a novel microchip-MS interface to enable sensitive MALDI-MS/MS detection directly from the chip. Specific Aim 2: Demonstrate the effectiveness of the microfluidic MALDI-MS/MS platform for the detection of multiple cancer biomarkers in cellular extracts. Cellular fractions from the MCF7 breast cancer cell line will be analyzed for target proteins that are involved in essential cellular processes driving cancer on-set and development (cell proliferation, cell cycle regulation, DNA repair, apoptosis and invasion/metastasis).